Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIβ

ABSTRACT Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses.

1. When talking about antiviral drugs, the readers always want to know the IC50 and CC50 of the drug. The authors should calculate the IC50 of vemurafenib against selected enteroviruses. 2. Fig.2C, RT-PCR data, please show the DNA bands in agarose gel. 3. Fig.3B, error bar was not shown? 4. Fig.5A, please indicate the molecular weight of the targeted proteins. 5. Fig.6, can the authors show the Western blot analysis of PI4KB in the increasing dose of vemurafenib? This is the most important data on mechanistic study.
Reviewer #2 (Comments for the Author): Laajala et al. reported an FDA-approved RAF kinase inhibitor vemurafenib potentially targets cellular PI4KB for anti-EV activity. Anti-EV-A71 activity of vemuferanib has been reported by Hu et al. in 2022. The potential importance of the current work might be in the target of vemurafenib for the anti-EV activity.
Some concerns on technical issues and the writing are as below.
Major points: 1. I could not agree that too many important references related to PI4KB and OSBP were missed in an awkward way. I would strongly suggest the authors completely correct this issue for the readership of this manuscript. 2. L35, Fig.1: Values of CC50(after short(7 h) and long (equal or more than 2 days) treatment), EC50, and selectivity index of vemuferanib for virus infection and of EC50 for killing of melanoma cells and normal cells should be provided and discussed on the potential potency as antiviral. Hu et al. already reported that the EC50 value of vemurafenib to EV-A71 infection was at the order of nM (Hu et al., 2022, Pharmaceuticals). 3. The labels in Fig.1C should be magnified. The current figure is invalid. 4. IC50 values of vemuferanib for BRAF (V600E/K) and PI4KB activities should be provided and discussed on the potential potency as above. Even if the inhibitory effect of vemuferanib on PI4KB activity could not be evaluated for any reason, this important point should be clarified as a limitation of this study to avoid misleading the readers. Most of the current discussion is based on a possible inference that vemuferanib is a PI4KB inhibitor. 5. L141, 152, L237: Conditions of viral infection are not clearly described; at least MOI should be described for all the viruses. 6. L128: An MOI of 400 seemed too high for evaluation of the antiviral effect against EV infection. An explanation of the condition may be included. 7. Fig.2C. Explanation of the gradual increment of the viral titer in Mock-treated cells should be added. 8. L356: Information on used drug library or the compounds should be provided. 9. L606: A c-RAF kinase inhibitor (GW5074) was known as a PI4KB inhibitor (Arita et al., 2008, JGV, 2011, JVI). This fact should be included here because this working hypothesis was not a novel one. 10. Fig.6A: Intracellular localization of PI4KB in OSW-1 and ITZ-treated cells was almost invisible. The current data set is not sufficient to show the effect of vemurafenib on the relocalization of Pi4KB. Clear data would be required. 11. Fig.6B: The effect of ITZ needs to be clarified. 12. Fig.6C: Observed resistance was partial, and possibly affected by the high cytotoxicity of vemurafenib. The statistical significance should be shown to support the resistance of the CVB3(H57Y) mutant. In addition, the cytotoxicity should be clearly discussed with the CC50 values in the used cells. 13. Fig. S6C. CC50 and EC50 values should be shown. 14. L724-740: The target population of the anti-EV drug, cytotoxicity in vitro, and side effects in vivo of vemuferanib should be clearly discussed in the context of the drug reposition. Vemuferanib is not applicable to young healthy children. 15. L746: Complete resistance of a poliovirus mutant against PI4KB/OSBP inhibitors has been reported (Arita and Bigay, 2019, ACS Infect Dis). 16. L717: The link between GBF1 and PI4KB accumulation has not been clarified to date.

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Point by point responses
Reviewer comments:

Reviewer #1 (Comments for the Author):
The manuscript by Laajala et al., examined the antiviral activity of vemurafenib against enteroviruses and several other DNA and RNA viruses. Specifically, they showed that Vemurafenib was effective against group B and C enteroviruses as well as rhinovirus but not parechovirus, Semliki Forest virus, adenovirus, or human respiratory syncytial virus. They also tried to understand the mechanism of action of this antiviral drug. They found that the antiviral effect of vemurafenib was due to the inhibitory of cellular PI4KB, but not RAF/MEK/ERK pathway. Overall, the study was well designed, the procedures were well described, and the results are interesting. The manuscript was also well-written and can be potentially published in this journal. However, several points should be addressed. These comments are: 1. When talking about antiviral drugs, the readers always want to know the IC50 and CC50 of the drug. The authors should calculate the IC50 of vemurafenib against selected enteroviruses.
We thank the reviewer for this suggestion. We have now added the IC50 values against selected enteroviruses ( Figure 1A) and CC50 (Figure 1B). Fig.2C, RT-PCR data, please show the DNA bands in agarose gel.

2.
We thank the reviewer for this suggestion. The gel image has now been added to Figure 2C.

Fig.3B, error bar was not shown?
We thank the reviewer for this notion. The error bars were accidentally left out from the graph, but they have now been added in Figure 3B. Fig.5A, please indicate the molecular weight of the targeted proteins.

4.
We have now added the molecular weights in Figure 5A. Fig.6, can the authors show the Western blot analysis of PI4KB in the increasing dose of vemurafenib? This is the most important data on mechanistic study. Figure 6A. Schaar et al. 2012). We have now also added this reference in the results section, page 21.

Reviewer #2 (Comments for the Author):
Laajala et al. reported an FDA-approved RAF kinase inhibitor vemurafenib potentially targets cellular PI4KB for anti-EV activity. Anti-EV-A71 activity of vemuferanib has been reported by Hu et al. in 2022. The potential importance of the current work might be in the target of vemurafenib for the anti-EV activity.
Some concerns on technical issues and the writing are as below.
Major points: 1. I could not agree that too many important references related to PI4KB and OSBP were missed in an awkward way. I would strongly suggest the authors completely correct this issue for the readership of this manuscript.
We thank the reviewer for this comment and apologize that there were not enough references concerning PI4KB and OSBP. We have now added many new references both in the results and discussion sections. Please see our more detailed comments below. Fig.1: Values of CC50(after short (7 h) and long (equal or more than 2 days) treatment), EC50, and selectivity index of vemuferanib for virus infection and of EC50 for killing of melanoma cells and normal cells should be provided and discussed on the potential potency as antiviral. Hu et al. already reported that the EC50 value of vemurafenib to EV-A71 infection was at the order of nM (Hu et al., 2022, Pharmaceuticals).

L35,
We thank the reviewer for this suggestion. We have now added the IC50 values of selected enteroviruses ( Figure 1A) and CC50 data of vemurafenib for short and long timepoints (Figure 1 B) as well as SI ( Figure 1B) in A549 cells. We have not done our studies on melanoma or normal cells as the idea was to study the effects in A549 cells lacking the V600 mutation. See also page 16. Fig.1C should be magnified. The current figure is invalid.

The labels in
We apologize that the labels in Fig.1C were not clearly visible. The figure has now been corrected.
4. IC50 values of vemuferanib for BRAF (V600E/K) and PI4KB activities should be provided and discussed on the potential potency as above. Even if the inhibitory effect of vemuferanib on PI4KB activity could not be evaluated for any reason, this important point should be clarified as a limitation of this study to avoid misleading the readers. Most of the current discussion is based on a possible inference that vemuferanib is a PI4KB inhibitor.
We thank the referee for the great suggestion. We were able to perform the activity assay on BRAF (V600E/K). This result is shown here below for the referee to see. There was only one vendor, Abcam, who originally suggested that they could provide PI4KB for activity testing. However, Abcam ran into trouble trying to make the product, and they continuously postponed the shipment, and, after waiting for several weeks, Abcam again notified about yet another long waiting time. Therefore, we decided to submit the revision now as there was no clear indication that we could get any product in the end. We are sorry for this. Therefore, we have added this as the limitation of the study as the referee suggested. Also, we feel that adding the BRAF activity alone is not needed information as we lack the PI4KB data. Please, see page 23-24.
5. L141, 152, L237: Conditions of viral infection are not clearly described; at least MOI should be described for all the viruses.
We thank the reviewer for this comment. In the rhinovirus, EV1 and HPEV1 assays we have used ice binding where the virus is attached to the receptor through 1h ice binding after which excess virus is thoroughly washed. Hence, determination of exact MOI is not possible, but instead, only the receptor bound virus is allowed to infect the cells.
In adenovirus assay, we have used MOI 30 and the info has been added in the methods section, page 7. 6. L128: An MOI of 400 seemed too high for evaluation of the antiviral effect against EV infection. An explanation of the condition may be included.
The reviewer is right, very high MOI was used here but still vemurafenib showed efficacy against the infection. However, as we have added the IC50 data of EV1 and CVA9 and all the CVB serotypes in Figure 1A, we have now removed this data.